title: The new beta-D-glucosidase in terpenoid-isoquinoline alkaloid biosynthesis in Psychotria ipecacuanha.
abstract: Ipecac alkaloids produced in the medicinal plant Psychotria ipecacuanha such as emetine and cephaeline possess a monoterpenoid-tetrahydroisoquinoline skeleton, which is formed by condensation of dopamine and secologanin. Deglucosylation of one of the condensed products N-deacetylisoipecoside (1 alpha(S)-epimer) is considered to be a part of the reactions for emetine biosynthesis, whereas its 1 beta(R)-epimer N-deacetylipecoside is converted to ipecoside in P. ipecacuanha. Here, we isolated a cDNA clone Ipeglu1 encoding Ipecac alkaloid beta-D-glucosidase from P. ipecacuanha. The deduced protein showed 54 and 48% identities to raucaffricine beta-glucosidase and strictosidine beta-glucosidase, respectively. Recombinant IpeGlu1 enzyme preferentially hydrolyzed glucosidic Ipecac alkaloids except for their lactams, but showed poor or no activity toward other substrates, including terpenoid-indole alkaloid glucosides. Liquid chromatography-tandem mass spectrometry analysis of deglucosylated products of N-deacetylisoipecoside revealed spontaneous transitions of the highly reactive aglycons, one of which was supposed to be the intermediate for emetine biosynthesis. IpeGlu1 activity was extremely poor toward 7-O-methyl and 6,7-O,O-dimethyl derivatives. However, 6-O-methyl derivatives were hydrolyzed as efficiently as non-methylated substrates, suggesting the possibility of 6-O-methylation prior to deglucosylation by IpeGlu1. In contrast to the strictosidine beta-glucosidase that stereospecifically hydrolyzes 3 alpha(S)-epimer in terpenoid-indole alkaloid biosynthesis, IpeGlu1 lacked stereospecificity for its substrates where 1 beta(R)-epimers were preferred to 1 alpha(S)-epimers, although ipecoside (1 beta(R)) is a major alkaloidal glucoside in P. ipecacuanha, suggesting the compartmentalization of IpeGlu1 from ipecoside. These facts have significant implications for distinct physiological roles of 1 alpha(S)- and 1 beta(R)-epimers and for the involvement of IpeGlu1 in the metabolic fate of both of them.
authors.affiliation: Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA. tnomura@danforthcenter.org
authors.forename: Taiji
authors.initials: T
authors.lastname: Nomura
authors.forename: Alfonso Lara
authors.initials: AL
authors.lastname: Quesada
authors.forename: Toni M
authors.initials: TM
authors.lastname: Kutchan
mesh_terms: D000470:Alkaloids / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D028503:Cephaelis / Q000201:enzymology* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007546:Isoquinolines / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry*; D001617:beta-Glucosidase / Q000737:chemistry*
chemical_list: D000470:Alkaloids; D018076:DNA, Complementary; D007546:Isoquinolines; D011994:Recombinant Proteins; D013729:Terpenes; D001617:beta-Glucosidase